With the advent of fluorescence probes targeting specific cellular and subcellular events, the role of fluorescent imaging in preclinical and clinicl arena is getting progressively stronger. Fluorescence probes mainly consist of the active component, which interacts with the target, and the reporting component such as the fluorescent dye. Operating in the near-infrared spectrum in which the tissue absorption is minimum, these probes can be detected in vivo even if they are located deep. The advantages of fluorescence imaging are its high sensitivity, low-cost and contrast agents with long shelf-life However, high tissue scattering leads to poor resolution and low quantitative accuracy that hinder the progress of fluorescence imaging towards clinical applications. An intriguing solution to this problem is to use a focused ultrasound beam and tag the diffused fluorescence photons in the focal zone. By detecting and analyzing the tagged (or modulated) fluorescence photons, distribution of the fluorescence probes can be recovered with higher resolution since ultrasound can be focused into a small zone deep in tissue. However, this technique suffers from the low signal to noise ratio due to limited modulation depth in the optical signal generated by the ultrasound modulation. Our main goal in this proposal is to use focused ultrasound to improve fluorescence imaging as well but in a different way: by achieving thermal modulation in the focal zone. Utilizing recently available temperature sensitive fluorescent molecular probes, our approach is thermal outlining using focused ultrasound (TOFU) and uses this as a priori information for fluorescence imaging to attain superior resolution and excellent quantitative accuracy. The advantage of our approach is the high optical modulation depth provided by these contrast agents (up to ten-fold) with only 4K temperature increase. We have already developed a 2D prototype system and in this study we will extend it to a 3D TOFU system. Once optimized with extensive phantom studies, we will evaluate the performance of the proposed technique with small animals bearing PSMA positive orthotopic tumor models together with PSMA targeting temperature sensitive fluorescent molecular probes.